I. The S100 Proteins
There are now more than 20 members of the S100 family of EF-hand Ca2+-binding proteins, which are known to be widely distributed in human tissue (Zimmer et al, Brain Res. Bull., 37:417-429 (1995); Donato, Int. J. Biochem. Cell. Biol., 33:637-668 (2001); and Heizmann et al, Frontiers in Bioscience 7:1356-1368 (2002)). S100 proteins were given this name because they are soluble in 100% saturated ammonium sulfate (Moore, Biochem. Biophys. Res. Comm., 19:739-744 (1965)). One member, S100B, is a 21.5 kDa symmetric homodimer that is highly conserved (>95%) among mammals (Zimmer et al, supra; and Moore, supra). In a manner similar to calmodulin, a Ca2+-dependent conformational change is required for S100B to bind a target protein (FIG. 1; Rustandi et al, Nat. Struct. Biol., 7:570-574 (2000); Rustandi et al, Biochem., 37:1951-1960 (1998); Weber et al, “Interaction of Dimeric S100B(ββ) with the Tumor Suppressor Protein: A Model for Ca2+-dependent S100-Target Protein Interactions”, The Molecular Basis of Calcium Action in Biology and Medicine (Pochet, R., Ed.), Kluwer Academic Publishers, Dordrecht, The Netherlands (2000); and Kligman et al, Trends Biochem. Sci., 13:437-443 (1988)).
In general, low levels of S100B have trophic effects, and higher levels are toxic, resulting in uncontrolled cell growth (Castets et al, Brain Res., 46:208-216 (1997); Van Eldik et al, Biochimica et Biophysica Acta, 1223:398-403 (1994); Mariggio et al, Neuroscience, 60:29-35 (1994); and McLendon et al, In: Cancer diagnosis in vitro using monoclonal antibodies (Kubchik, H. Z., ed) Vol. 39, pp. 31-66, Marcel Dekker, New York (1988). Increased levels of S100B are found in renal cell tumors (Takashi et al., Urol. Res., 22:251-255 (1994)), and malignant mature T-cells (such as doubly negative CD4−/CD8− adult T-cells in leukemia patients) (Suzushima et al, Leuk. Lymph., 13:257-262 (1994)). Furthermore, S100B is up regulated by other cytokines that stimulate gliosis, such as interleukin-1β and the basic fibroblast growth factor (Hinkle et al, Neuroscience, 82:33-41 (1998)).
As is the case for S100B, a number of other S100 proteins are regulated in a tissue-specific manner (Kligman et al, supra). S100A1, calcyclin (S100A6), and S100B levels are elevated significantly in metastatic human mammary epithelial cells (Pedrocchi et al, Int. J. Cancer, 57:684-690 (1994)), and increased levels of S100A4 mouse (mts1) in transgenic mice induce metastatic mammary tumors (Chen et al, J. Biol. Chem., 272:20283-20290 (1997)). In the case of mts1, protein levels are controlled in benign cell lines via a cis-acting element 1300 base pairs upstream of the rat mts1 start site (Chen et al, supra), and expression of antisense RNA to mts1 suppresses metastatic potential for a high-metastatic Lewis lung carcinoma (Takenaga et al, Oncogene, 14:331-337 (1997)). Protein levels of S100B, mts1, and calcyclin correlate with malignant melanoma. Thus, S100 proteins are used as markers for this cancer (Maelandsmo et al, Int. J. Cancer, 74:464-469 (1997); Boni et al, J. Cutan. Pathol., 24:76-80 (1997); Xia et al, Cancer Res., 57:3055-3062 (1997); Hansson et al, Anticancer Res., 17:3071-3073 (1997); and FIG. 2B).
S100 antibodies are used clinically to identify and classify malignant tumors in several tissues including brain, lung, bladder, intestine, kidney, cervix, breast, skin, head and neck, lymph, testes, larynx, and mouth among others (Takashi et al, supra; Suzushima et al, supra; Pedrocchie et al, supra; Fisher et al, J. Clin. Path., 47:868-869 (1994); Iniue et al, J. Urol., 85:495-503 (1994); Kerrebijn et al, Cancer Immun. Immunother., 38:31-37 (1994); Colasante et al, Am. Rev. Resp. Dis., 148:752-759 (1993); Zeid et al, Path., 25:338-343 (1993); Gallo et al, Arch. Otolarn., 117:1001-1010 (1991); Wilson et al, J. Path., 163:25-30 (1991); Lee et al; Proc. Natl. Acad. Sci. USA, 89:2504-2508 (1992); Leong et al, J. Path., 162:35-41 (1990); Nakano et al, Arch. Path. Lab. Med., 113:507-511 (1989); Kurihara et al, J. Oral. Path., 14:289-298 (1985); Matsushima et al, J. Surg. Onc., 55:108-113 (1994); Renshaw et al, Mod. Path., 10:693-700 (1997); Larock et al, Vet. Path., 34:303-311 (1997); and Hurley et al, J. Med. Primat., 26:172-180 (1997)).
II. p53
p53 is a transcription activator that signals for cell cycle arrest and apoptosis and plays a pivotal role in the maintenance and regulation of normal cellular functions (Levine et al, Nature, 351:453-456 (1991); and Levine, Cell, 88:323-331 (1997)). The inactivation of p53 affects cell cycle checkpoints, apoptosis, gene amplification, centrosome duplication and ploidy (Levine (1997), supra; Woods et al, Exp. Cell. Res., 264:56-66 (2001); Burns et al, J. Cell. Physiol., 181:231-239 (2001); Appella et al, Eur. J. Biochem., 268:2764-2772 (2001); Arrowsmith et al, Cell Death Differ., 6:1169-1173 (1999); Prives et al, J. Pathol., 187:112-126 (1999); Vousden, Cell, 103:691-694 (2000); and Ryan et al, Curr. Opin. Cell. Biol., 13:332-337 (2001)). If p53 is inactivated by mutation, as found in over 50% of human cancers, the cell cycle proceeds unregulated and cell growth proliferates. Likewise, apoptosis pathways are not induced, and proliferating cells transform into cancerous ones (Woods et al, supra; Burns, supra; Appella et al, supra; Arrowsmith et al, supra; Prives et al (1999), supra; Vousden, supra; Ryan et al, supra; and Agarwal et al, J. Biol. Chem., 273:1-4 (1998)). On the other hand, if p53 levels are too high, then problems associated with aging occur (Tyner et al, Nature, 415:45-53 (2002)). p53 is highly regulated by post-translational modifications and by interactions with other proteins inside the cell (Appella et al, supra; Minamoto et al, Oncogene, 20:3341-3347 (2001); Jayaraman et al, Cell. Mol. Life Sci., 55:76-87 (1999); Jimenez et al, Oncogene 18:7656-7665 (1999); and Meek, Pathol. Biol., 45:804-814 (1997)).
III. The S100-p53 Interaction
S100B and several other S100 proteins (i.e., S100A1 and mts1) interact with the tumor suppressor protein, p53, in cancer cells resulting in significantly reduced p53 levels, and p53-dependent transcription activation of target genes is inhibited (Grigorian et al, J. Biol. Chem., 276:22699-22708 (2001); Lin et al, J. Biol. Chem., 276:35037-35041 (2001); and Carrier et al, Proc. AACR, 40:102 (1999)).
The interactions between the C-terminus of p53 and S100 calcium binding proteins, such as S100B are of particular interest since like p53, S100 proteins affect cell cycle progression, are over expressed in numerous tumor cells, and are associated with tumor progression (Ilg et al, Int. J. Cancer, 68:325-332 (1996)). These results correlate with knowledge that p53 and S100B (i) interact tightly in vitro (KD=24±10 nM; Delphin et al, J. Biol. Chem., 274:10539-10544 (1999)), (ii) S100B inhibits PKC-dependent phosphorylation of p53 (Wilder et al, Protein Sci., 7:794-798 (1998); and Baudier et al, Proc. Natl. Acad. Sci., USA, 89:11627-11631 (1992)), (iii) S100B dissociates the p53 tetramer (Baudier et al, supra), (iv) subunits of both S100B and the C-terminus of p53 associate via an X-type four-helix bundle structural motif (Weber et al, supra; Jeffrey et al, supra; and Lee et al, supra); and (v) three S100 proteins (S100B, S100A1, mts1) inhibit p53 function in vivo (Grigorian et al, supra; Lin et al, supra; and Carrier et al (1999), supra).
In the present invention it is demonstrated that in primary malignant melanoma cancer cells, S100B interacts directly with p53 in a cell-cycle dependent manner resulting in lower levels of wild-type p53. In addition, it was found in the present invention that the S100B promoter has three sequences that bind to p53, which supports the notion that S100B transcription, in turn, is regulated by p53 in a feed back loop that triggers p53's own degradation.